library(ArchR)
## Loading required package: ggplot2
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archProj <- loadArchRProject("../data/scATAC/archR_subHybridClusters/",
                 showLogo = FALSE)
## Successfully loaded ArchRProject!
plotEmbedding(ArchRProj = archProj, colorBy = "cellColData", 
              name = "predAnn", embedding = "UMAP_cor")
## ArchR logging to : ArchRLogs/ArchR-plotEmbedding-1051127c59bbd-Date-2021-10-22_Time-11-12-16.log
## If there is an issue, please report to github with logFile!
## Getting UMAP Embedding
## ColorBy = cellColData
## Plotting Embedding
## 1 
## ArchR logging successful to : ArchRLogs/ArchR-plotEmbedding-1051127c59bbd-Date-2021-10-22_Time-11-12-16.log

archProj <- addMotifAnnotations(ArchRProj = archProj, 
                                motifSet = "cisbp", 
                                name = "Motif",
                                force=TRUE)
## No methods found in package 'IRanges' for request: 'score' when loading 'TFBSTools'
## ArchR logging to : ArchRLogs/ArchR-addMotifAnnotations-105111e73a9b1-Date-2021-10-22_Time-11-12-19.log
## If there is an issue, please report to github with logFile!
## peakAnnotation name already exists! Overriding.
## 2021-10-22 11:12:23 : Gettting Motif Set, Species : Mus musculus, 0.002 mins elapsed.
## Using version 2 motifs!
## 2021-10-22 11:12:26 : Finding Motif Positions with motifmatchr!, 0.042 mins elapsed.
## 2021-10-22 11:15:28 : Creating Motif Overlap Matrix, 3.084 mins elapsed.
## 2021-10-22 11:15:32 : Finished Getting Motif Info!, 3.147 mins elapsed.
## ArchR logging successful to : ArchRLogs/ArchR-addMotifAnnotations-105111e73a9b1-Date-2021-10-22_Time-11-12-19.log
## get ArchR peak markers for motif enrichment
markerPeaks_subHybrid <- getMarkerFeatures(
    ArchRProj = archProj, 
    useMatrix = "PeakMatrix", 
    groupBy = "predAnn",
    bias = c("TSSEnrichment", "log10(nFrags)"),
    testMethod = "wilcoxon")
## ArchR logging to : ArchRLogs/ArchR-getMarkerFeatures-105113b89b315-Date-2021-10-22_Time-11-15-36.log
## If there is an issue, please report to github with logFile!
## MatrixClass = Sparse.Integer.Matrix
## 2021-10-22 11:15:36 : Matching Known Biases, 0.004 mins elapsed.
## 2021-10-22 11:15:37 : Computing Pairwise Tests (1 of 5), 0.026 mins elapsed.
## 2021-10-22 11:16:00 : Computing Pairwise Tests (2 of 5), 0.399 mins elapsed.
## 2021-10-22 11:16:22 : Computing Pairwise Tests (3 of 5), 0.769 mins elapsed.
## 2021-10-22 11:16:43 : Computing Pairwise Tests (4 of 5), 1.115 mins elapsed.
## 2021-10-22 11:17:03 : Computing Pairwise Tests (5 of 5), 1.459 mins elapsed.
## ###########
## 2021-10-22 11:17:26 : Completed Pairwise Tests, 1.837 mins elapsed.
## ###########
## ArchR logging successful to : ArchRLogs/ArchR-getMarkerFeatures-105113b89b315-Date-2021-10-22_Time-11-15-36.log
peakMarkers_subHybrid <- getMarkers(markerPeaks_subHybrid, 
                                    cutOff = "FDR <= 0.01 & Log2FC >= 1.25", 
                                    returnGR = TRUE)
heatmapMarkerPeaks <- plotMarkerHeatmap(
  seMarker = markerPeaks_subHybrid, 
  cutOff = "FDR <= 0.01 & Log2FC >= 1.25", 
  transpose = TRUE
)
## ArchR logging to : ArchRLogs/ArchR-plotMarkerHeatmap-105114d4c463c-Date-2021-10-22_Time-11-17-29.log
## If there is an issue, please report to github with logFile!
## Identified 20694 markers!
## Preparing Main Heatmap..
## ArchR logging successful to : ArchRLogs/ArchR-plotMarkerHeatmap-105114d4c463c-Date-2021-10-22_Time-11-17-29.log
ComplexHeatmap::draw(heatmapMarkerPeaks, heatmap_legend_side = "bot", annotation_legend_side = "bot")

Check accessibility of Egfr target genes

egfrTargetDf <- read.csv("../data/genesTargets/egfr_targets_unfiltered.csv")
pCluster <- plotEmbedding(ArchRProj = archProj, 
                    colorBy = "cellColData", 
                    name = "predAnn", 
                    embedding = "UMAP_cor")
## ArchR logging to : ArchRLogs/ArchR-plotEmbedding-105113423f8f7-Date-2021-10-22_Time-11-17-36.log
## If there is an issue, please report to github with logFile!
## Getting UMAP Embedding
## ColorBy = cellColData
## Plotting Embedding
## 1 
## ArchR logging successful to : ArchRLogs/ArchR-plotEmbedding-105113423f8f7-Date-2021-10-22_Time-11-17-36.log
pCluster

allGenes <- unname(unique(mcols(archProj@geneAnnotation$genes)$symbol))
egfrTargets <- intersect(as.character(egfrTargetDf$target), allGenes)

## get peak count matrix
ArrowFiles <- getArrowFiles(archProj)
featureDF <- ArchR:::.getFeatureDF(head(ArrowFiles, 2), "PeakMatrix")
peakMat <- ArchR:::.getPartialMatrix(ArrowFiles, 
            featureDF = featureDF, threads = 1, useMatrix = "PeakMatrix", 
            cellNames = rownames(archProj@cellColData), progress = FALSE)
## 2021-10-22 11:17:38 : Getting Partial Matrix 1 of 6, 0 mins elapsed.
## 2021-10-22 11:17:46 : Getting Partial Matrix 2 of 6, 0.141 mins elapsed.
## 2021-10-22 11:17:49 : Getting Partial Matrix 3 of 6, 0.187 mins elapsed.
## 2021-10-22 11:17:52 : Getting Partial Matrix 4 of 6, 0.249 mins elapsed.
## 2021-10-22 11:17:57 : Getting Partial Matrix 5 of 6, 0.318 mins elapsed.
## 2021-10-22 11:18:00 : Getting Partial Matrix 6 of 6, 0.368 mins elapsed.
## 2021-10-22 11:18:08 : Successfully Created Partial Matrix, 0.504 mins elapsed.
## for each gene, get the peak closest to TSS
peakGR <- archProj@peakSet
tssPeaks <- c()
for(gg in 1:length(egfrTargets)){
  curGene <- egfrTargets[gg]
  curID <- which(mcols(peakGR)$nearestGene == curGene)
  if(length(curID) > 0 ){
    curTSSPeak <- curID[which.min(mcols(peakGR)$distToTSS[curID])]
    tssPeaks[gg] <- curTSSPeak
  } else {
    tssPeaks[gg] <- NA
    next
  }
}

egfrTargets <- egfrTargets[!is.na(tssPeaks)]
tssPeaks <- tssPeaks[!is.na(tssPeaks)]
egfrTargetPeaks <- peakMat[tssPeaks,]
# egfrTargetPeakScaledSum <- colSums(t(scale(t(egfrTargetPeaks))))
egfrTargetPeakScaledSum <- colSums(egfrTargetPeaks)


umapEmbedding <- getEmbedding(archProj, embedding="UMAP_cor")
colnames(umapEmbedding) <- paste0("UMAP",1:2)
umapEmbedding$egfrTargetScaledSum <- egfrTargetPeakScaledSum

pEgfrTargets <- ggplot(umapEmbedding, aes(x=UMAP1,
                          y=UMAP2,
                          col=egfrTargetScaledSum)) +
  theme_classic() +
  geom_point() +
  scale_color_gradientn(colors=wesanderson::wes_palette("Zissou1", n=100, type="continuous"))

pEgfrTargets

Check accessibility of peaks containining Ets TFBS motif

motifMatches <- readRDS(archProj@peakAnnotation$Motif$Matches)
etsMotifId <- grep(x=colnames(motifMatches), pattern="Ets")
etsMotifs <- grep(x=colnames(motifMatches), pattern="Ets", value=TRUE)
peaksWithEtsMotifs <- which(rowSums(assays(motifMatches[,etsMotifs])$matches) > 0)
length(peaksWithEtsMotifs)
## [1] 10594
umapEmbedding$etsMotifPeaks <- colSums(peakMat[peaksWithEtsMotifs,])

pEtsMotifs <- ggplot(umapEmbedding, aes(x=UMAP1,
                          y=UMAP2,
                          col=etsMotifPeaks)) +
  theme_classic() +
  geom_point() +
  scale_color_gradientn(colors=wesanderson::wes_palette("Zissou1", n=100, type="continuous"))

pEtsMotifs

Association between Egfr targets’ accessibility and Ets motif peaks’ accessibility

plot(umapEmbedding$egfrTargetScaledSum, umapEmbedding$etsMotifPeaks,
     xlab = "Egfr targets' accessibility",
     ylab = "Ets-motif-containing peaks' accessibility")

Gene markers for (sub)hybrid clusters

plotEmbedding(ArchRProj = archProj, colorBy = "cellColData", 
              name = "predAnn", embedding = "UMAP_cor")
## ArchR logging to : ArchRLogs/ArchR-plotEmbedding-10511521f7928-Date-2021-10-22_Time-11-18-13.log
## If there is an issue, please report to github with logFile!
## Getting UMAP Embedding
## ColorBy = cellColData
## Plotting Embedding
## 1 
## ArchR logging successful to : ArchRLogs/ArchR-plotEmbedding-10511521f7928-Date-2021-10-22_Time-11-18-13.log

## get ArchR gene markers for motif enrichment
markerGenes_subHybrid <- getMarkerFeatures(
    ArchRProj = archProj, 
    useMatrix = "GeneScoreMatrix", 
    groupBy = "predAnn",
    bias = c("TSSEnrichment", "log10(nFrags)"),
    testMethod = "wilcoxon")
## ArchR logging to : ArchRLogs/ArchR-getMarkerFeatures-105114351803c-Date-2021-10-22_Time-11-18-15.log
## If there is an issue, please report to github with logFile!
## MatrixClass = Sparse.Double.Matrix
## 2021-10-22 11:18:15 : Matching Known Biases, 0.001 mins elapsed.
## 2021-10-22 11:18:16 : Computing Pairwise Tests (1 of 5), 0.02 mins elapsed.
## 2021-10-22 11:18:37 : Computing Pairwise Tests (2 of 5), 0.371 mins elapsed.
## 2021-10-22 11:19:01 : Computing Pairwise Tests (3 of 5), 0.768 mins elapsed.
## 2021-10-22 11:19:23 : Computing Pairwise Tests (4 of 5), 1.128 mins elapsed.
## 2021-10-22 11:19:42 : Computing Pairwise Tests (5 of 5), 1.454 mins elapsed.
## ###########
## 2021-10-22 11:20:03 : Completed Pairwise Tests, 1.79 mins elapsed.
## ###########
## ArchR logging successful to : ArchRLogs/ArchR-getMarkerFeatures-105114351803c-Date-2021-10-22_Time-11-18-15.log
geneMarkers_subHybrid <- getMarkers(markerGenes_subHybrid, 
                                    cutOff = "FDR <= 0.01 & Log2FC >= 1.25")

write.csv(geneMarkers_subHybrid$C2_Hybrid, file="../data/scATAC/markerGenesHybrid.csv")
write.csv(geneMarkers_subHybrid$hybrid1, file="../data/scATAC/markerGenesHybrid1.csv")
write.csv(geneMarkers_subHybrid$hybrid2, file="../data/scATAC/markerGenesHybrid2.csv")
write.csv(geneMarkers_subHybrid$C1_Inj, file="../data/scATAC/markerGenesInjured.csv")
write.csv(geneMarkers_subHybrid$resting, file="../data/scATAC/markerGenesResting.csv")

Marker genes for hybrid1

markerGenesHybrid1 <- geneMarkers_subHybrid$hybrid1$name[1:18]
p <- plotEmbedding(
    ArchRProj = archProj, 
    colorBy = "GeneScoreMatrix", 
    name = markerGenesHybrid1, 
    embedding = "UMAP_cor",
    quantCut = c(0.01, 0.95),
    size=1/3,
    plotAs="points"
)
## Getting ImputeWeights
## No imputeWeights found, returning NULL
## ArchR logging to : ArchRLogs/ArchR-plotEmbedding-1051151ca2bc5-Date-2021-10-22_Time-11-20-03.log
## If there is an issue, please report to github with logFile!
## Getting UMAP Embedding
## ColorBy = GeneScoreMatrix
## Getting Matrix Values...
## 2021-10-22 11:20:03 :
## 1 2 3 4 5 6 
## Plotting Embedding
## 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 
## ArchR logging successful to : ArchRLogs/ArchR-plotEmbedding-1051151ca2bc5-Date-2021-10-22_Time-11-20-03.log
# clean
p2Hybrid1 <- lapply(p, function(x){
    x + guides(color = FALSE, fill = FALSE) + 
    theme_ArchR(baseSize = 6.5) +
    theme(plot.margin = unit(c(0, 0, 0, 0), "cm")) +
    theme(
        axis.text.x=element_blank(), 
        axis.ticks.x=element_blank(), 
        axis.text.y=element_blank(), 
        axis.ticks.y=element_blank()
    )
})
cowplot::plot_grid(plotlist=p2Hybrid1[1:9])

cowplot::plot_grid(plotlist=p2Hybrid1[10:18])

Marker genes for hybrid2

markerGenesHybrid2 <- geneMarkers_subHybrid$hybrid2$name[1:18]
p <- plotEmbedding(
    ArchRProj = archProj, 
    colorBy = "GeneScoreMatrix", 
    name = markerGenesHybrid2, 
    embedding = "UMAP_cor",
    quantCut = c(0.01, 0.95),
    size=1/3,
    plotAs="points"
)
## Getting ImputeWeights
## No imputeWeights found, returning NULL
## ArchR logging to : ArchRLogs/ArchR-plotEmbedding-105113eadff98-Date-2021-10-22_Time-11-20-24.log
## If there is an issue, please report to github with logFile!
## Getting UMAP Embedding
## ColorBy = GeneScoreMatrix
## Getting Matrix Values...
## 2021-10-22 11:20:25 :
## 1 2 3 4 5 6 
## Plotting Embedding
## 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 
## ArchR logging successful to : ArchRLogs/ArchR-plotEmbedding-105113eadff98-Date-2021-10-22_Time-11-20-24.log
# clean
p2Hybrid2 <- lapply(p, function(x){
    x + guides(color = FALSE, fill = FALSE) + 
    theme_ArchR(baseSize = 6.5) +
    theme(plot.margin = unit(c(0, 0, 0, 0), "cm")) +
    theme(
        axis.text.x=element_blank(), 
        axis.ticks.x=element_blank(), 
        axis.text.y=element_blank(), 
        axis.ticks.y=element_blank()
    )
})
cowplot::plot_grid(plotlist=p2Hybrid2[1:9])

cowplot::plot_grid(plotlist=p2Hybrid2[10:18])

Marker genes for other hybrid cells

markerGenesHybrid <- geneMarkers_subHybrid$C2_Hybrid$name[1:18]
p <- plotEmbedding(
    ArchRProj = archProj, 
    colorBy = "GeneScoreMatrix", 
    name = markerGenesHybrid, 
    embedding = "UMAP_cor",
    quantCut = c(0.01, 0.95),
    size=1/3,
    plotAs="points"
)
## Getting ImputeWeights
## No imputeWeights found, returning NULL
## ArchR logging to : ArchRLogs/ArchR-plotEmbedding-105111f04b5cf-Date-2021-10-22_Time-11-20-44.log
## If there is an issue, please report to github with logFile!
## Getting UMAP Embedding
## ColorBy = GeneScoreMatrix
## Getting Matrix Values...
## 2021-10-22 11:20:45 :
## 1 2 3 4 5 6 
## Plotting Embedding
## 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 
## ArchR logging successful to : ArchRLogs/ArchR-plotEmbedding-105111f04b5cf-Date-2021-10-22_Time-11-20-44.log
# clean
p2Hybrid <- lapply(p, function(x){
    x + guides(color = FALSE, fill = FALSE) + 
    theme_ArchR(baseSize = 6.5) +
    theme(plot.margin = unit(c(0, 0, 0, 0), "cm")) +
    theme(
        axis.text.x=element_blank(), 
        axis.ticks.x=element_blank(), 
        axis.text.y=element_blank(), 
        axis.ticks.y=element_blank()
    )
})
cowplot::plot_grid(plotlist=p2Hybrid[1:9])

cowplot::plot_grid(plotlist=p2Hybrid[10:18])